Though enzymatic activity has long been considered the exclusive domain of proteins, discoveries in molecular biology over the past couple of decades have led to the realization that ribonucleic acid (RNA) can also function as an enzyme. RNA enzymes are often referred to as ribozymes.
Ribozyme substrates are generally confined to RNA molecules, and enzymatic activities of ribozymes include the cleavage and/or ligation of RNA molecules. The cleavage activity may be intramolecular, known as cis-acting or intermolecular, known as trans-acting. There are at least five classes of ribozymes known, including Group I introns, Group II introns, hammerhead, hairpin, and delta ribozymes. The last three are derived from plant satellites and viroids.
Since 1982, several unexpected diseases caused by RNA-based pathogenic agents have emerged. These include the lethal Acquired Immune Deficiency Syndrome (AIDS) and delta hepatitis, a particularly virulent form of fulminant hepatitis caused by a viroid-like RNA agent. These blood-borne diseases are spread at the RNA level, manifest themselves in cells of patients, and are by now present within the bloodstream of millions of individuals. Conventional biotechnology, with its reliance on recombinant DNA methods and DNA-level intervention schemes, has been slow to provide valid approaches to combat these diseases.
Two forms of delta ribozymes, namely genomic and antigenomic, are derived, and referred to by, the polarity of the hepatitis delta virus (HDV) genome from which the ribozyme is generated. Like hammerhead and hairpin ribozymes, the delta ribozymes cleave a phosphodiester bond of their RNA substrates and give rise to reaction products containing a 5′-hydroxyl and a 2′,3′-cyclic phosphate termini. They are metalloenzymes and a low concentration (<1 mM) of magnesium (Mg2+) or calcium (Ca2+) is required for delta ribozyme cleavage. Both genomic strand and antigenomic strand forms exhibit self-cleavage activity, and it has been suggested that they are involved in the process of viral replication (Lazinski, D. W., and Taylor, J. M. (1995) RNA 1, 225–233).
Delta ribozymes derived from the genome of HDV are of interest in the development of a gene regulation system in which the designed ribozymes would down-regulate the expression of a target gene. The facts that delta ribozymes are derived from HDV and that this pathogen naturally replicates in animal systems, suggest that this catalytic RNA could be used to control gene expression in human cells. Like other ribozymes, the designed ribozyme should specifically cleave its target substrates while leaving other cellular RNA molecules intact.
Trans-acting ribozymes carry out intermolecular cleavage activity. Some trans-acting delta ribozymes have been developed by removing a single-stranded junction which connects the catalytic portion to the substrate portion in cis-acting delta ribozymes. This results in two separate molecules, one possessing the substrate sequence and the other the catalytic property (Been, M. D. and Wichhan, G. S. (1997) Eur. J. Biochem., 247, 741–753). Interactions between such delta ribozymes and the substrate occur through the formation of a helix, referred as the P1 stem. However, the example of the trans-acting ribozyme disclosed by Been et al. (supra) was not useful for cleaving long substrate molecules, such as those having therapeutic applications.
In U.S. Pat. No. 5,225,337, issued on Jul. 6, 1993 in the names of Hugh D. Robertson et al., there are disclosed ribozymes derived from a specific domain present in the HDV RNA for specifically cleaving targeted RNA sequences and uses thereof for the treatment of disease conditions which involve RNA expression, such as AIDS. These ribozymes consist of at least 18 consecutive nucleotides from the conserved region of HDV isolates between residues 611 and 771 on the genomic strand and between residues 845 and 980 on the complementary antigenomic strand. These ribozymes are proposed to fold into an axe-head model secondary structure (Branch, A. D., and Robertson, H. D. (1991) Proc. Natl. Acad. Sci. USA 88, 10163–10167). The ribozymes developed according to this model structure require the substrate to be bound to the ribozyme through the formation of two helices, one located on either side of the cleavage site. Further, such ribozymes apparently require a 12–15 nucleotide recognition sequence in the substrate in order to exhibit the desired activity. Such a long recognition sequence is not practical in the development of therapeutic or diagnostic applications.
In U.S. Pat. No. 5,625,047, issued on Apr. 29, 1997 in the names of Michael D. Been et al., there are disclosed enzymatic RNA molecules proposed to fold into a pseudoknot model secondary structure (discussed below). The method disclosed for the development of efficient ribozymes requires a short recognition sequence of only 7 to 8 nucleotides in the substrate, a preference for a guanosine base immediately 3′ to the cleavage site, a preference for U, C or A immediately 5′ to the cleavage site, and the availability of a 2′-hydroxyl group for cleavage to occur. Thus, the specificity of recognition of these ribozymes is limited to 6 or 7 base pairing nucleotides with the substrate and a preference of the first nucleotide located 5′ to the cleavage site. Neither tertiary interaction(s) between the base paired nucleotides and another region of the ribozyme, nor single-stranded nucleotides are involved to define the specificity of recognition of these ribozymes. Because the recognition features are limited, these ribozymes have a limited specificity, and thus, are not practical for further clinical or biotechnical applications.
A pseudoknot-like structure for delta ribozymes has been proposed by Perrotta and Been (Perrotta, A. T., and Been, M. D. (1991) Nature 350, 434–436). This model structure consists of two stems (P1 and P2), two stem-loops (P3 and P4) and three single-stranded regions (J1/2, J1/4 and J4/2). An additional stem, named P1.1, has been formed by two GC base pairs between nucleotides from the J1/4 junction and the P3 loop (Ferré-D'Amaré, A. R., Zhou, K. and Doudna, J. A. (1998) Nature, 350, 434–436).
It would be highly desirable to be provided with a novel delta ribozyme for the cleavage of both small and large RNA substrates for which the specificity of recognition is well defined. Such specificity would yield optimal conditions for further therapeutical and biotechnological developments of delta ribozymes.